DESCRIPTION (Adapted from abstract): In all cells, the site of protein synthesis is the ribosome, and its importance is reflected in the many drugs and antibiotics that are produced to disrupt its operation. To understand how the ribosome efficiently and accurately synthesizes proteins according to the genetic blueprint, it is important to determine its structure. For such a complex object, it has been necessary to combine structural information from several biochemical and biophysical techniques. The approach taken in this proposal is to analyze the structures of crucial ribosomal components by x-ray crystallography and NMR spectroscopy, and to use these structures as high-resolution probes of the ribonucleoprotein assembly. The overall goal of this research has been, and continues to be, to understand the mechanism of protein synthesis at the molecular level. A major focus of the proposal is to study the complexes of 30S subunit primary RNA-binding ribosomal proteins with fragments of cognate RNA. The structures of the majority of these proteins in the absence of RNA have already been determined. Significant progress has been made for proteins S4, S7, S8, and S15 for which the crystal structures are known, and preliminary crystals of the S7-RNA complex have been obtained. Future studies will include the S17-RNA complex using both x-ray and NMR methods since the S17 NMR structure has previously been determined. In parallel, it is proposed to begin construction of a model of the 30S subunit using current structures of the 16S rRNA and ribosomal proteins, and future structures of protein-RNA complexes. Two important sites within the large 50S subunit will also be studied. Excellent crystals have been grown of the L11-RNA complex that is central to the GTPase center, and the structures of proteins L2 and L3 from the peptidyl transferase center will be determined. Finally, work will continue on the ribosomal initiation complex and initiation factor IF3. Cryo-electron microscopy has been used to produce an image of the 30S-IF3 complex in which the IF3 crystal structure can be recognized. Future work will improve this image to accurately orient the IF3 molecule, and additional factors will be added to build up the complete initiation complex.